全 文 ：Received 5 Aug. 2003 Accepted 29 Aug. 2003
Supported by the National Natural Science Foundation of China (30070059).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Assessment of Genetic Variation and Distribution Pattern of
Thalictrum petaloideum Detected by RAPDs
XIE Lei, LI Liang-Qian*, ZHANG Da-Ming
(Laboratory of Systematic and Evolutionary Botany, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China)
Abstract: Random amplified polymorphic DNA (RAPD) method was applied to assess genetic variation
and population structure of Thalictrum petaloideum L. (Ranunculaceae). Two hundred and forty-six
individuals from 11 populations of the species were investigated by RAPD profiles. Twenty selected RAPD
primers generated 125 bands, in which 120 were polymorphic. The results revealed a high level of genetic
variation (percentage of polymorphic bands (PPB) was 96%, Nei’s gene diversity (h) was 0.350 2 and
shannon’s information index (I ) was 0.519 9 at the species level). The differentiation among the
populations was high (GST = 0.351 1) in this species. Result of analyzing of molecular variance (AMOVA)
showed that 38.88% of genetic variance was found among the populations. Positive correlation with r =
0.194 5 (P = 0.000 2) was found between genetic distance and geographic distance among populations. Two
populations distributed in the drainage basin of Yangtz River affined genetically and formed one clade and
the rest nine populations formed the other clade in both unweighted pair-group method using arithmetic
average (UPGMA) trees made by two different methods. It was very clear that these two populations were
very special, and must be closely related in history, despite the fact that they now share quite weak link to
the rest populations through gene communication.
Key words: Thalictrum petaloideum ; random amplified polymorphic DNA (RAPD); genetic diversity;
distribution pattern
The genus Thalictrum is one of the largest and most
diverse genera in buttercup family (Ranunculaceae).
Thalictrum petaloideum is a species of the genus, which is
a diploid and widely distributed in northern China with sev-
eral populations reaching the drainage basin of Yangtz River
(Wu and Raven, 2001) (Fig.1). Plants in the drainage basin
of Yangtz River have some characters different from the
ones in northern China, marked by bigger leaflets, stronger
stems, shadowy and lower altitude inhabited environment.
Wang (1984) considered that its modern distribution pat-
tern might be affected by the glacial events and its final
geographical structure was most likely formed through re-
colonizing after the last glacial events and in Pleistocene
Ice Age. In this study, The random amplified polymorphic
DNA (RAPD) (Williams et al., 1990) method was used to
analyze the population genetic structure of T. petaloideum
and provided valuable molecular evidence for its genetic
variation.
Population genetic structure is considered as spatio-
temporal distribution of genetic variation (Ge, 1997). It can
reflect evolutionary processes of populations (Hamrick and
Loveless, 1989). In order to elucidate these processes, pat-
terns and mechanisms of evolution and to explain
phylogenetic relationships among taxa, we should reveal
the range of genetic variation, genetic structure, diversifica-
tion trend and other factors affecting genetic structure of
the populations.
Presently, many biochemical and DNA molecular mark-
ers have been used in the studying of population structure.
Fig.1. Distributional pattern of Thalictrum petaloideum.
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 165-170
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004166
RAPD is frequently applied to reveal population genetic
variation, divergence and biogeography (Schaal and
Leverich, 2001). A large number of studies have success-
fully applied this method to study genetic variation in plants
despite its shortcomings, such as unnecessity of prior DNA
sequence information, relatively unbiased portion of the
genome sampling, simplicity to use, lower cost, and smaller
amount of plant material used (Qian et al., 2000).
1 Materials and Methods
1.1 Materials
Leaves of Thalictrum petaloideum L. collected from 246
individuals of 11 populations and dried by silica gel were
stored at –20 ℃ (Table 1). Individuals of nine populations
that covered most of northern distribution area of T.
petaloideum were involved and two populations from the
drainage basin of Yangtz River were also carefully gathered
for the study.
1.2 Methods
1.2.1 DNA isolation Total genomic DNA of a single leaf
was extracted from silica-gel-dried leaves with a modified
CTAB method (Zou et al., 2001).
1.2.2 RAPD-PCR amplification Twenty of 100 primers
provided by Operon, Shanghai, which produced clear and
polymorphic banding patterns among different populations
in the prior screening were selected (A-15, B-5, D-20, G-8,
G-10, G-19, L-18, S-18, T-6, V-6, Y-11, Y-14, Y-20, Z-17, Z-18,
K-9, K-10, O-15, P-2, R-1). PCR reactions were carried out in
a volume of 10 mL containing 10 ng template DNA, 50 mmol/L
Tris-HCL（pH 8.3）, 500 mg/mL BSA，10% Ficoll, 1
mmol/L Tartrazine, 2 mmol/L MgCl2，200 mmol/L dNTP, 1
mmol/L primer and 0.5 U Taq polymerase. DNA amplifica-
tion was performed in a Rapidcycler 1818 (Idaho Tech.),
programmed for an initial 94 ℃ 60 s, 36 ℃ 10 s, 72 ℃ 20 s
for 2 cycles, followed by 40 cycles of 94 ℃ 0 s, 36 ℃ 0 s,
and 72 ℃ 60 s, and ended with 72 ℃ 7 min. Amplification
products were analyzed by electrophoresis on 1.5% agar-
ose gel stained with ethidium bromide, and photographed
under ultraviolet light. Molecular weights were estimated
using a 100 bp DNA ladder.
1.2.3 Data analysis Amplified fragments were scored as
binominal data, i.e., the presence as 1 and absence as 0 for
the homologous bands. The matrix of RAPD phenotype
was analyzed based on several indices of population
genetics, such as observed number of alleles (na), effec-
tive number of alleles (ne), Nei’s gene diversity (h),
shannon’s information index (I) and GST through POPGENE
program (Yeh et al., 1997). DCFA1.0 (Zhang, 2001) program
was also used to calculate the similarity coefficients for all
pairs of samples. We then analyzed the distribution of ge-
netic variance by Analysis of Molecular Variance
（AMOVA）（Excoffier，1993）for combining the
similarity coefficients. Two dendrograms using unweighted
pair group method (UPGMA) (Sneath and Sokal, 1973) were
constructed for estimating the genetic similarity based on
Nei’s coefficients and average taxonomic distance of band
frequency. NTSYSpc2.02a software（Rohlf，1997）was
used to calculate the correlation between genetic distance
and geographic distance among populations.
2 Results
2.1 Level of polymorphism
Twenty decamer primers produced 125 reproducible and
clear amplification bands, which were employed for analyz-
ing (Fig. 2).
We got 245 RAPD phenotypes from 246 individuals of
T. petaloideum. The percentage of polymorphic bands
(PPB) at species level was 96.00% (Table 2).
2.2 Population genetic structure
Four indices of population genetics: observed number
of alleles (na), effective number of alleles (ne), Nei’s gene
diversity (h), shannon’s information index (I), were shown
Table 1 Origin and sampling size of Thalictrum petaloideum used in this study
Population Origin Date Voucher Habitat
Altitude Sample
(m) size
YKS Yakeshi, Nei Mongol Jun., 2002 XL200251 Mountaintop; near cropland 700 23
QS Qianshan, Liaoning Jul., 2002 XL200266 Sunny mountaintop 1 000 22
BHS Baihuashan, Beijing May, 2002 XL200201 Meadow of the mountaintop 1 900 27
X W Xiaowutaishan, Hebei Jun., 2002 XL200246 Sunny and dry place 1 400 22
W T Wutaishan, Shanxi Jun., 2002 W200206 Sunny slope of the mountain 1 800 16
ZX Zheyangshan, Gansu Jul., 2002 XL200294 Sunny and dry meadow 2 200 21
TS Tianshui, Gansu Jul., 2002 XL200288 Sunny and dry 2 000 27
XJ Tianshan, Xinjiang Jun., 2002 XL200277 Mountain slope; meadow 1 500 20
BQ Baoku, Qinghai Sept.2002 XL200275 Sunny mountain slope 2 850 24
HB Zaoyang, Hubei Jun., 2002 XL200234 Forest shade 330 24
LY Langyashan, Anhui May,2002 XL200224 Forest shade 150 21
167XIE Lei et al.: Assessment of Genetic Variation and Distribution Pattern of Thalictrum petaloideum Detected by RAPDs
Fig.2. Amplified RAPD electrophorestic pattern of Thalictrum
petaloideum. a. Primer p178. b. Primer p90. c. Primer p291.
Table 2 Percentage of polymorphic bands (PPB) in different
populations of Thalictrum petaloideum
Population Sample size
Number of
PPB (%)
polymorphic bands
YKS 23 78 62.40
QS 21 67 53.60
BHS 27 90 72.00
X W 22 78 62.40
W T 16 70 56.00
ZX 21 62 49.60
TS 27 69 55.20
BQ 24 72 57.60
XJ 20 89 71.20
HB 24 76 60.80
LY 21 71 56.80
Average 22.4 76.9 61.53
Species 246 120 96
Abbrevations are the same as in Table 1.
variation are distributed within populations (Table 4).
2.4 UPGMA results
Two UPGMA dendrograms, both illustrating the genetic
relationships among populations, were identical in topol-
ogy (Figs.3, 4).
We used NTSYSpc2.02a software（Rohlf，1997）
to test the correlation between genetic distance and geo-
graphic distance among populations by its MXCOMP
function. The correlation between two similarity matrices
showed a positive correlation with r = 0.194 5 with high
in Table 3. GST computed by POPGENE is 0.351 1, and after
excluding the two Yangtz River populations, GST is 0.301 9.
2.3 Results of AMOVA analyses
Results of AMOVA analyses indicated that majority of
Table 3 Indices of population genetics in Thalictrum petaloideum
Population Population size
Observed number of Effective number of Nei’s gene Shannon’s information
alleles (na) alleles (ne) diversity (h) index (I)
YKS 23 1.624 0 1.408 6 0.232 2 0.343 1
QS 21 1.536 0 1.335 7 0.190 3 0.282 2
BHS 27 1.720 0 1.468 4 0.262 0 0.385 7
X W 22 1.624 0 1.445 9 0.248 1 0.361 2
W T 16 1.520 0 1.337 7 0.193 1 0.285 7
ZX 21 1.496 0 1.346 8 0.196 6 0.287 8
TS 27 1.552 0 1.376 4 0.214 6 0.314 4
XJ 20 1.712 0 1.489 9 0.273 3 0.399 7
BQ 24 1.576 0 1.317 9 0.190 4 0.288 4
HB 24 1.608 0 1.376 6 0.215 4 0.319 6
LY 21 1.568 0 1.359 1 0.204 9 0.303 6
Average 22.4 1.680 0 1.455 0 0.258 0 0.380 0
Variance 0.054 8 0.039 8 0.023 2 0.034 0
Species 246 1.984 0 1.609 5 0.350 2 0.519 9
Abbreviations are the same as in Table 1.
Table 4 Results of Analysis of Molecular Variance (AMOVA) analysis in Thalictrum petaloideum
Source of variation Variance expectation Percentage P value
Among populations 8.348 8 38.88% <0.01
Within population 13.127 0 61.12% <0.01
Significance tests were performed using 1 000 permutations.
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004168
statistic significance (P = 0.000 2).
3 Discussion
3.1 Genetic diversity and population structure
We examined the level of genetic polymorphism in T.
petaloideum by RAPD analysis. At species level, the per-
centage of polymorphic bands (PPB) were 96%, it was higher
than that of other species reported before in the
Ranunculaceae ① (Markus et al. , 2000; Cole and
Kuchenreuther，2001). Shannon’s information index (I)
showed the same result, and its value of 0.519 9 was much
higher than the other results of population-level studies.
Nei’s gene diversity（h）gotten in our research was
0.350 2. According to Nybom and Bartish (2000), average
value of dicotyledons was 0.191. It also indicated that high
level of genetic polymorphism indwelled this species. This
may attribute to its outcrossing reproductive behavior and
lacking of clonal vegetation.
The GST value of the species was 0.351 1, meaning that
35.11 per cent of the total variation was among populations.
This value was rather high (Hamrick and Godt, 1990). It
indicated that distinctive divergence happened among
populations. Theoretically, higher GST values would exist
in annual, incrossing and spreading seeds by weight, herba-
ceous species than that in perennial, outcrossing and
spreading seeds by wind species. As for T. petaloideum,
this result seemed to be conflicting and its story must be
different. It was perennial herb, but the life span of indi-
viduals was relatively short. Moreover, although it was a
widely distributed species in northern China, the distribu-
tion area was rather discontinuous. Based on our fieldwork,
individuals of T. petaloideum distributed in northern area
were restricted in higher altitude, sunny and dry places. So
discontinuity of environment resulted in isolating of
populations. Furthermore, distance of the populations, as
represented by our sampling, was long enough to avoid
intercommunication of gene among populations. These
were the main causes of the high value of the GST. AMOVA
analyses also showed much inter-population variation. From
what was discussed above we could find that T.
petaloideum was a species that possessed abundant varia-
tion and is under the process to form new subspecies.
3.2 Geographical inferences
Two UPGMA dendrograms showed identical topology.
Two populations from the drainage basin of Yangtz River
were clustered together and separated from the other nine
populations from the root. The other nine northern popula-
tions formed two clades, which represented the plants in
eastern and western areas respectively. We tested the cor-
relation between genetic and geographic distances ( r =
0.194 5). It suggested that those two factors were posi-
tively related although the correlation was weak. This was
because the two Yangtz River populations had greater ge-
netic distance and smaller geographic distance from the
other nine populations. When these two populations were
removed, the value from the rest nine populations was rather
high (0.541 6). From these results, we knew that apart from
Yangtz River populations, distinctive relationship between
the genetic distance and the geographical distance could
be found. Whereas, the two Yangtz River populations ex-
hibited high genetic similarity but they were distinctive from
the northern populations. Although to draw a conclusion
might not be safe on how these two populations had
Fig.3. Diagram of UPGMA dendrograms made by average
taxonomic distance of band frequency illustrating the genetic rela-
tionships among populations in Thalictrum petaloideum.
Abbreviatioins are the same as in Table 1.
Fig.4. Diagram of UPGMA dendrograms made by Nei’s coeffi-
cients illustrating genetic relationships among populations in
Thalictrum petaloideum. Abbreviations are the same as in Table 1.
① Zhang F-M. 2002. A preliminary study on speciation of Ac-
onitum delavayvi complex (Ranunculaceae) in Hengduan
Mountains. PhD thesis.
169XIE Lei et al.: Assessment of Genetic Variation and Distribution Pattern of Thalictrum petaloideum Detected by RAPDs
proceeded a evolutionary process in developing and di-
versifying from the rest northern populations yet from the
RAPD data, it was very clear that these two populations
were very special, and these two ones must be closely re-
lated in history, despite the fact that they now share quite
weak link to the rest populations through gene
communication.
Acknowledgements: Thanks go to Dr. LIU Jian-Quan, Dr.
WANG Ying-Wei and Mr. MAO Jian-Feng for sampling
from Qinghai, Shanxi and Xinjiang. We would also like to
thank Prof. GE Song for his excellent comments on the
manuscript. We were indebted to Dr. ZHANG Fu-Min for
his kindly instruction on data processing.
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